Assessing Chemical Constituents of Mimosa caesalpiniifolia Stem Bark: Possible Bioactive Components Accountable for the Cytotoxic Effect of M. caesalpiniifolia on Human Tumour Cell Lines

Mimosa caesalpiniifolia is a native plant of the Brazilian northeast, and few studies have investigated its chemical composition and biological significance. This work describes the identification of the first chemical constituents in the ethanolic extract and fractions of M. caesalpiniifolia stem bark based on NMR, GC-qMS and HRMS analyses, as well as an assessment of their cytotoxic activity. GC-qMS analysis showed fatty acid derivatives, triterpenes and steroid substances and confirmed the identity of the chemical compounds isolated from the hexane fraction. Metabolite biodiversity in M. caesalpiniifolia stem bark revealed the differentiated accumulation of pentacyclic triterpenic acids, with a high content of betulinic acid and minor amounts of 3-oxo and 3β-acetoxy derivatives. Bioactive analysis based on total phenolic and flavonoid content showed a high amount of these compounds in the ethanolic extract, and ESI-(−)-LTQ-Orbitrap-MS identified caffeoyl hexose at high intensity, as well as the presence of phenolic acids and flavonoids. Furthermore, the evaluation of the ethanolic extract and fractions, including betulinic acid, against colon (HCT-116), ovarian (OVCAR-8) and glioblastoma (SF-295) tumour cell lines showed that the crude extract, hexane and dichloromethane fractions possessed moderate to high inhibitory activity, which may be related to the abundance of betulinic acid. The phytochemical and biological study of M. caesalpiniifolia stem bark thus revealed a new alternative source of antitumour compounds, possibly made effective by the presence of betulinic acid and by chemical co-synergism with other compounds.


Introduction
Mimosa L. (Fabaceae) is the second largest genus of the Mimosoideae subfamily and comprises approximately 530 species, distributed mainly in South and Central America [1][2][3]. Despite the great biodiversity of the genus Mimosa, phytochemical and pharmacological studies are restricted to about twenty representatives, of which Mimosa pudica and Mimosa tenuiflora are the most commonly investigated species.
Mimosa caesalpiniifolia Benth. (syn. Mimosa caesalpiniaefolia) is a native plant to northeastern Brazil, popularly known as "unha-de-gato", "sabiá", "angiquinho-sabiá" and "sansão do campo" [19,20]. Due to their therapeutic effects, the stem bark and flowers have been used in traditional medicine for the treatment of bronchitis, skin infections and injuries [21] and for inflammation and hypertension [22], respectively. Therefore, detailed phytochemical studies are necessary to identify the active compound(s) for phytotherapeutic product development and to possibly aid in the search for new drugs. Taking into account previous chemical and pharmacological studies of M. caesalpiniifolia [23,24] and screenings for the antiproliferative activity of Brazilian species, this work describes the first phytochemical study of M. caesalpiniifolia stem bark. Non-polar compounds and acid derivatives of hexane and dichloromethane fractions were analysed by GC-qMS, and polar compounds from ethanolic extract were identified by HRMS and MS n experiments. Additionally, ethanolic extract, fractions and isolated compounds were evaluated for cytotoxic activity against the HCT-116, OVCAR-8 and SF-295 human tumour cell lines.

Results and Discussion
The extraction of M. caesalpiniifolia stem bark yielded a crude ethanolic extract with 4.2% (w/w) extractable matter. Partitioning of the EtOH extract of M. caesalpiniifolia with different solvents yielded fractions at extraction efficiencies ranging from 7.4% to 37.7%. The aqueous fraction had the highest extraction efficiency (37.7%, w/w), whereas the n-hexane fraction presented the lowest extraction efficiency (7.4%, w/w). The efficiency of fractionation for different fractions in descending order was as follows: aqueous > dichloromethane > ethyl acetate > hexane.
The 1 H-and 13 C-NMR analysis of subfraction F4 showed signals similar to a 3β-hydroxy-lup-20(29)-ene skeleton, except for the presence of a signal at δC 181.4, which indicated a carbonyl group in C-28 and the absence of δC 18.1 (CH3). The total ion chromatogram showed one peak for the methylated sample ( Figure S1

Non-Polar Compounds and Acid Derivatives of M. caesalpiniifolia by GC-qMS
Analysis of the chemical composition of extracts, fractions and natural compounds by GC-qMS is a useful tool in phytochemical studies for the separation and identification of individual compounds from organic matrices, especially in bioactivity-guided studies [28]. To determine the phytoconstituents and fatty acids of M. caesalpiniifolia stem bark, the hexane and dichloromethane fractions were methylated using diazomethane solution. The GC-qMS profiles of the methylated hexane and dichloromethane fractions from the ethanolic extract of M. caesalpiniifolia are shown in Figure 2.  Table 1.  Compound identification in the GC-qMS analysis was performed using retention time and interpretation of the mass spectra (molecular ion [M +• ], base peak and main fragments) in comparison with mass spectra of isolated compounds, computational libraries and literature data [29][30][31][32].
In addition, the presence of triterpenic acid derivatives (13.17% to 71.54%) indicated the great metabolic biodiversity of M. caesalpiniifolia, mostly the high accumulation of betulinic acid (3β-hydroxy-lup-20(29)-en-28-oic acid) in the stem bark. The triterpenic acid derivatives showed m/z 262 for the acid methyl ester group at C-28, except in olean-18-ene acid methyl ester, which showed m/z 248. In the urs-12-ene acid methyl ester skeleton, an additional fragment at m/z 133 (base peak) was observed [29,30].
On the other hand, α-tocopherol was the single phenolic compound detected in the hexane fraction (relative intensity 0.02%) using the GC-qMS extracted ion chromatogram (EIC), at m/z 165. The fragment at m/z 165 [C10H13O2] + resulted from hydrogen rearrangement and retro-Diels-Alder cleavage of the pyran ring ( Figures S2 and S3, Supplementary Materials). In comparative chromatograms, α-tocopherol showed a relative abundance on the order of 100 times less than methyl octacosanoate.
The presence of phenolic compounds in the dichloromethane fraction was detected using EIC m/z 77, 91 and 105, which correspond to related benzene ring compounds, and m/z 94 and 108 for phenols, including a base peak at m/z 272 [M +• ] indicative of C6-C3-C6 derivatives, in accordance with the NIST mass library. However, phenolic compounds in the dichloromethane fraction were found at less than 0.05% (trace level), which complicates their identification due to poor-quality spectra.

Total Phenolic and Flavonoid Contents of M. caesalpiniifolia Stem Bark
The stem bark of M. caesalpiniifolia is characterised by the common occurrence of polyphenols and tannins (water-soluble phenolic compounds) [33]. The importance of polyphenols and related compounds in Mimosa species is associated with biological and pharmacological properties, such as antioxidant, cytotoxic and antimicrobial activity. Phytochemical investigation of Mimosa invisa showed the presence of phenolic substances, which include flavonoids, flavonoid glycosides and lignans with promising antiradical activity (e.g., quercetin, a well-known natural antioxidant) [5]. Two phenolic compounds (a deoxyflavone derivative and flavolignan) isolated from Mimosa diplotricha were active in an antiproliferative assay against tumour cell lines [4]. The cinnamic acid diterpenyl ester isolated from Mimosa pudica was highly active against the microorganisms Malassezia pachydermatis, Candida albicans and Staphylococcus aureus [34].
The total phenolic content (TPC) and total flavonoid content (TFC) in the ethanol extract and fractions of M. caesalpiniifolia are shown in Table 2. TPC values are expressed in milligrams of gallic acid equivalent per gram of dried plant material (mg GAE/g DPM), determined by the Folin-Ciocalteu method using gallic acid as a standard. TFC values are expressed in milligrams of rutin equivalent per gram of dried plant material (mg RE/g DPM), determined by the aluminium complex method using a rutin analytical curve. The ethanolic extract had the highest total phenolic content (14.8 mg GAE/g DPM), followed by the aqueous fraction (6.42 mg GAE/g DPM), whereas the hexane fraction had the lowest content (0.16 mg GAE/g DPM). It was observed that the phenolic content of the extract and fractions of M. caesalpiniifolia obeyed a decreasing order: ethanol > aqueous > ethyl acetate > dichloromethane > hexane. Phenolic compounds in the hexane fraction were confirmed by GC-qMS analysis, which showed a lower accumulation of α-tocopherol in M. caesalpiniifolia stem bark (Section 2.1.1.).
In the present investigation, the total flavonoid content (TFC) ranged from 0.34 to 1.81 mg RE/g DPM. The ethanol extract had the highest TFC (1.81 mg RE/g DPM), followed by the aqueous extract with 0.68 mg RE/g DPM. TFC extracted in solvents with different polarity were found to obey the following decreasing order: ethanol > aqueous > dichloromethane ≥ ethyl acetate (p < 0.05, one-way ANOVA). Generally, flavonoids are rarely found in extractions and partitioning with low-polarity solvents such as n-hexane [35]. According to GC-qMS analysis, flavonoids and C6-C3-C6 derivatives were not detected in the hexane fraction. Therefore, phenolic acids, polyphenol flavonoid-like compounds and tannins in the ethanolic extract of M. caesalpiniifolia stem bark were investigated by ESI(−)-MS.

Identification of Phenolic Compounds in M. caesalpiniifolia by ESI(−)-LTQ-Orbitrap-MS
In this study, several phenolic compounds were tentatively identified in the ethanolic extract of M. caesalpiniifolia stem bark using negative electrospray ionisation coupled to a linear ion trap-orbitrap hybrid mass spectrometer (ESI(−)-LTQ-Orbitrap-MS) in the scan mode and multi-stage mass analysis (MS n ) (Figure 3). This method provides molecular and structural information for chemical identification. The phenolic composition of ethanolic extract was analysed because this extract had the highest concentration of total phenols and flavonoids, 14.8 mg GAE/g DPM and 1.81 mg RE/g DPM (Table 2)   ethanolic extract, the plant contained a high intensity of ions at m/z 377.08 and 341.11, which confirms a high content of caffeoyl hexose in M. caesalpiniifolia stem bark.

Cytotoxic Activity
The ethanolic extract, partition fractions (hexane, dichloromethane, ethyl acetate and aqueous), betulinic acid and doxorubicin (positive control) were evaluated by MTT assay against colon (HCT-116), ovarian (OVCAR-8) and glioblastoma (SF-295) tumour cell lines. The positive control doxorubicin (at 0.3 µg/mL), a well-known anticancer drug that is widely used in clinical therapy [40], showed an inhibition of >83.0% for all tumour cell lines. The percentage inhibition of cell proliferation indicates that ethanolic extract and fractions showed moderate and high cytotoxic effects, with the exception of the ethyl acetate and aqueous fractions, as shown in Figure 4. The results showed that the dichloromethane fraction and betulinic acid were the most active (>75.0%), with inhibition of cell proliferation above 86.5%, whereas the ethanolic extract and hexane fraction varied from 69.5% to 84.8% and 65.5% to 86.4%, respectively. These results demonstrated that the composition of the extract and fractions contains compounds that may contribute to chemical specificity and ability to interfere on the grown tumour cells, as observed in previous cytotoxicity studies of plant extracts and fractions [41].
As mentioned above, the effectiveness of inhibition is clearly affected by the chemical composition of the extract and fractions, which may be responsible for additive, synergetic, or antagonistic effects. Our results demonstrate that the hexane fraction showed decreased activity against the glioblastoma tumour cell strain (SF-295) when compared with ethanolic extract, with significantly different values (p < 0.05). In contrast, despite the low activity of the ethyl acetate and aqueous fractions, these fractions exhibited a strong growth inhibition effect in SF-295 tumour cells, when compared with the inhibition effects in the other cell strains. In the specific case of HCT-116 tumour cells, the negative percent inhibition observed for the ethyl acetate fraction showed that there was cell growth, i.e., the fraction was inactive. The selectivity of the ethanolic extract and ethyl acetate and aqueous fractions against SF-295 tumour cells when compared with the hexane fraction is most likely due to the amount of polyphenol compounds, as shown in Table 2.
The ethanolic extract and hexane fraction showed no significant differences (one-way ANOVA, p > 0.05) for the inhibition of OVCAR-8 tumour cells. The one-way ANOVA test, applied to the data describing the inhibition of cell proliferation for the dichloromethane fraction and betulinic acid, showed that differences were not significant (p > 0.05) for all lines tested. In this context, the dichloromethane fraction and betulinic acid showed similarly powerful responses in the inhibition of the proliferation of neoplasic cells.
The cytotoxicity results might be related with the presence of betulinic acid, which was previously isolated from the hexane fraction (subfraction F4) and identified by GC-qMS in the dichloromethane fraction with a relative abundance of 70.30%. Literature, reports have shown the cytotoxic potential of betulinic acid [42,43]. Therefore, this study demonstrated that betulinic acid is the active compound responsible for the cytotoxic activity of M. caesalpiniifolia stem bark against tumour lines tested by the MTT assay.
It is important to highlight that the crude extract, which generally consists of a mixture of natural products such as phenolic acids, flavonoids, isoprenoids and primary metabolites such as fatty acid derivatives, was evaluated because it represents the most common approach in ethnopharmacological uses by Brazilian population. Moreover, it is likely that distinct bioactive molecules may jointly or independently contribute to the biological effects of plants [44,45]. Further studies are in progress to confirm these biological effects of M. caesalpiniifolia extracts.

Plant Material
The stem bark of M. caesalpiniifolia was collected in Teresina, Piauí, Brazil in May 2010. The plant specimen was identified and deposited in the Graziela Barroso Herbarium with voucher specimen number TEBP 26,824.

Analysis of Non-Polar Fractions of M. caesalpiniifolia by Gas Chromatography-Quadrupole Mass Spectrometry (GC-qMS)
The chemical constituents in the n-hexane and dichloromethane fractions were analysed by GC-qMS (GC7890A/VLMSD5975 system, Agilent Technologies) equipped with a DB-5 capillary column (J&W, 5% phenyl-95% methylpolysiloxane, 30 m × 250 mm × 0.25 μm). Samples were dissolved in n-hexane-EtOAc (1:1, v/v), after which previously prepared diazomethane diethyl ether solution was added. Sample aliquots of the solution (1 µL at 5 mg/mL w/v) were injected into a gas chromatograph in split mode (10:1). Helium was used as the carrier gas at a constant flow rate of 1 mL/min. The injector temperature was set to 310 °C. GC oven temperature program: initial temperature 150 °C (12 min); ramp at 4 °C/min to 290 °C (23 min). A mass spectrometer with quadrupole analyser was used, with electron ionisation (EI) at 70 eV, ion source at 300 °C, solvent delay time 8 min and mass range scan 40-650 Da. Chemical constituents were identified through the comparison of the obtained mass spectra with Wiley229 and NIST 0.8 computational libraries and authentic standards.

Total Phenol Content
Total phenol content (TPC) in the ethanol extract and fractions was measured using the Folin-Ciocalteu method described by Sousa et al. [46], with some modifications. Aliquots of dried samples dissolved in methanol (0.1 mL, 1000 μg/mL) were transferred to 10-mL volumetric flasks, followed by the addition of Folin-Ciocalteu reagent (0.5 mL) and distilled water (5 mL), and then samples were mixed for 1 min. Sodium carbonate (2 mL, 15% w/v) was added, and samples were stirred for 30 s. The volumetric flasks were filled to 10 mL with distilled water and incubated at room temperature for 2 h. The absorbance was measured at 750 nm using a UV-Vis spectrophotometer, and TPC content was determined in milligrams of gallic acid equivalent per gram of dry plant material (mg GAE/g DPM) using a gallic acid analytical curve (0.1-2.5 μg/mL, R = 0.999). All analyses were performed in triplicate (n = 3).

Total Flavonoid Content
Total flavonoid content (TFC) in the ethanol extract and fractions was determined using the aluminium complex method and a rutin analytical curve as described by Ferreira et al. [34]. Aliquots of dried samples dissolved in methanol (0.3 mL, 1000 μg/mL) were transferred to 10 mL volumetric flasks, followed by the addition of acetic acid (0.24 mL), pyridine in methanol (4 mL, 20% v/v) and aluminium chloride methanolic solution (1 mL, 5% w/v). The volumetric flasks were filled to 10 mL with distilled water. These solutions were incubated at room temperature for 30 min, and the absorbance was measured at 420 nm using a UV-Vis spectrophotometer. TFC contents were expressed in milligrams of rutin equivalent per gram of dry plant material (mg RE/g DPM) using a rutin analytical curve (3.0-21.0 μg/mL, R = 0.999). All analyses were performed in triplicate (n = 3).
For polyphenol analysis, a LTQ Orbitrap mass spectrometer was equipped with an ESI source in negative ionisation mode. FTMS mass spectra were acquired at a resolution of 100,000 in the mass range m/z 100 to 1000 Da on the Orbitrap analyser. The MS full mode parameters were as follows: spray voltage 3.30 kV; sheath gas 8 (arbitrary units); capillary voltage −46 V; capillary temperature 300 °C; and tube lens −115.14 V. The multi-stage analysis (MS n ) mode on the ion trap analyser for selected precursor ions was acquired by CID fragmentation using a normalised collision energy of 35.0. The data were processed using XCalibur software (version 2.1. Thermo Fischer Scientific Inc., San Jose, CA, USA), which provides possible elemental molecular formulas, accurate masses and isotopic patterns.

Evaluation of Cell Proliferation by MTT Assay
The cytotoxicity of ethanolic extract; the n-hexane, dichloromethane, ethyl acetate and aqueous fractions; and betulinic acid (isolated compound) and doxorubicin (positive control) was investigated by MTT assay [47], against HCT-116, OVCAR-8 and SF-295 cancer cells. This method analyses tumour cell growth by the ability of living cells to reduce the yellow dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) to a blue formazan product. Briefly, cells (0.1 × 10 6 cells/well) were plated in 96-well plates and incubated for 72 h with a medium consisting of DMSO (control group); ethanol extract, fractions and subfractions at 50.0 μg/mL; or doxorubicin (0.3 μg/mL, positive control). Then, the supernatant was replaced by fresh medium containing 0.15 mL of MTT, and the cells were incubated for an additional 3 h. The plates were centrifuged, the formazan product was dissolved in DMSO and the absorbance was measured at 595 nm using a multiplate reader (DTX 880 Multimode Detector, Beckman Coulter Inc., Fullerton, CA, USA). The percentage inhibition of cell growth was calculated, according to Mahmoud et al. [41], using the software GraphPad Prism (GraphPad Software, Inc., La Jolla, CA, USA). The experiment was performed in triplicate, and the results were expressed as the mean ± standard deviation (SD).

Statistical Analysis
A statistical approach was designed and experimental data were evaluated using one-way analysis of variance (ANOVA) using the software Origin 8.0 (OriginLab Corporation, Northampton, MA, USA), with a significance level of p < 0.05.